PROJECT ABSTRACT Hematopoietic stem cells (HSCs) are defined by their ability to long-term reconstitute all blood and immune cells in a transplanted host. However, HSCs are heterogeneous and exhibit different degrees of reconstitution potential and biases towards certain cell lineages. This diversity in HSCs is a function of both intrinsic changes in the cells, such as acquired somatic mutations and epigenetic marks, and external influences such as local bone marrow morphogens and circulating factors in the blood. It is evident from the transplantation of low numbers of purified HSCs that there exist at least two types of HSCs distinguishable by their contribution to different hematopoietic lineages?myeloid-biased HSCs, which contribute more to granulocytes and monocytes than to T, B, and NK cells, and balanced HSCs, which contribute equally to all lineages. During aging, the myeloid-biased subset of HSCs clonally expands and predominates the old bone marrow, leading to impaired blood and immune production and an increased risk of leukemia and preleukemic adverse events. Still, the origin and identity of the myeloid-biased HSCs, much less a method to prospectively separate them from balanced HSCs, is unknown. Recently, we observed that Neogenin-1 (Neo-1), a cell surface receptor, marks a fraction of long-term reconstituting mouse Hoxb5+ HSCs and human CD34+CD38-CD90+CD45RA- HSCs. Serial transplantation of Neo-1+ and Neo-1- Hoxb5+ HSCs into sublethally irradiated hosts reveals that Neo-1 marks stable, myeloid-biased mouse Hoxb5+ HSCs, while Neo-1- Hoxb5+ HSCs are balanced or lymphoid-biased. Neo- 1+ Hoxb5+ HSCs also formed larger colonies in vitro and were more often in G2/S than Neo-1- Hoxb5+ HSCs. Furthermore, Netrin-1, a chemoattractant molecule and ligand to Neo-1, is expressed in bone marrow endothelial cells and its depletion in mice results in reduced number of myeloid cells in the peripheral blood compared to wildtype controls. Given this evidence, two specific aims are proposed: (1) To identify the functional and transcriptional differences between Neo-1+ and Neo-1- HSCs during mouse and human aging, and (2) To determine the bone marrow localization and characterize the myeloid-priming function of endothelial Netrin-1. Percent expansion and gene expression differences will be measured between Neo-1+ and Neo-1- HSCs in mice and humans with age. Aged Neo-1+ and Neo-1- Hoxb5+ HSCs will be transplanted into conditioned mice and their total and lineage chimerism will be compared to our data from young Neo-1+ and Neo-1- Hoxb5+ transplants. Furthermore, bone marrow from Hoxb5-mCherry mice will be imaged to localize Netrin-1-expressing cells, and the effect of Netrin-1 deletion in endothelial cells will be evaluated. These studies will provide novel information on HSC heterogeneity and stem cell reconstitution and advance our understanding of aging in hematopoiesis.